Development and qualification of an enzyme-linked immunosorbent assay to detect human serum immunoglobulin G reactive to multiple lineages of Lassa virus nucleoprotein

Abstract

Lassa fever is a severe, often fatal febrile illness endemic to West Africa caused by Lassa virus (LASV). The viral nucleoprotein (NP) is a target antigen for serological assays to identify previous exposure to LASV. To our knowledge, there is no commercially available assay that reliably quantifies anti-LASV-NP IgG antibodies in human serum. We report the development and qualification of an ELISA designed to detect and quantify anti-LASV-NP IgG in human serum samples. The assay employs recombinant Lineage IV LASV-NP immobilized on microwells to capture NP-specific IgG antibodies, which are then detected using horseradish peroxidase-conjugated anti-human IgG followed by TMB substrate and reading of optical densities. Optimal assay reagent concentrations, incubation times and temperature were determined along with assay positivity criteria and dynamic range. A reference standard prepared from pooled sera from donors in endemic Lassa fever regions was established and calibrated to the first WHO international standard for LASV antibodies. High and low positive controls for assay quality control were generated. Naive human serum was used as a negative control. Following assay optimization, performance was assessed through assay qualification. The assay positivity criteria, lower limit of detection, upper and lower limits of quantitation, inter-assay precision, selectivity and dilutional linearity were determined. Our anti-LASV-NP IgG ELISA was shown to reliably measure anti-LASV-NP IgG levels in human serum. The method demonstrated sensitivity, intra- and inter-assay precision, dilution linearity across its analytical range and specificity for anti-LASV-NP IgG. Establishing this assay represents an essential step toward strengthening LASV epidemiology research and supporting urgently needed development of a vaccine to prevent Lassa Fever.